JP2006052145A - TNF-alpha INHIBITOR - Google Patents
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本発明はＴＮＦ−α阻害剤に関する。 The present invention relates to a TNF-α inhibitor.
ＴＮＦ−α（Tumor Necrosis Factor-α：腫瘍壊死因子）は、炎症を通した生体防御・免疫機構に広く関わるサイトカインとして認識されている（例えば、特許文献１参照。）。そして、ＴＮＦ−αの過剰な産生が組織障害やさまざまな病気（例えば、関節リウマチ、全身性エリテマトーデス（ＳＬＥ）、悪液質、急性感染症、アレルギー、発熱、貧血、糖尿病など。）を引き起こす原因となることが知られている（例えば、非特許文献１参照。）。また、ＴＮＦ−αが自己免疫性疾患である慢性関節リウマチおよびクローン病の発症に重要な役割を果たしていることも知られている（例えば、非特許文献２参照。）。 TNF-α (Tumor Necrosis Factor-α: tumor necrosis factor) is recognized as a cytokine widely involved in biological defense / immune mechanisms through inflammation (see, for example, Patent Document 1). And excessive production of TNF-α causes tissue damage and various diseases (for example, rheumatoid arthritis, systemic lupus erythematosus (SLE), cachexia, acute infection, allergy, fever, anemia, diabetes, etc.). (For example, see Non-Patent Document 1). It is also known that TNF-α plays an important role in the development of rheumatoid arthritis and Crohn's disease, which are autoimmune diseases (see, for example, Non-Patent Document 2).
このようなことから、ＴＮＦ−α阻害剤は、上記した疾患の治療に有効であると期待され、様々な化合物（例えば、６−フェニルテトラヒドロ−１，３−オキサジン−２−オン誘導体、側鎖に硫黄原子を有するウレア誘導体、ベンゾヘテロ環化合物、１，３−チアゾール化合物、メタロプロテアーゼ阻害剤、シクロスポリンＡなど。）についてのＴＮＦ−α阻害活性が研究されている（例えば、非特許文献１および２ならびに特許文献１〜７参照。）。
なお、ＴＮＦ−α阻害剤は、疼痛に対しても優れた予防・治療効果を発揮し、また、破骨細胞の活性化を抑制し、破骨細胞の形成を阻害することも知られている（例えば、特許文献６参照。）。また、ＴＮＦ−α阻害剤は、緑内障の治療にも使用できることが知られている（例えば、特許文献７参照。）。
For these reasons, TNF-α inhibitors are expected to be effective for the treatment of the above-mentioned diseases, and various compounds (for example, 6-phenyltetrahydro-1,3-oxazin-2-one derivatives, side chains) TNF-α inhibitory activity on urea derivatives having a sulfur atom, benzoheterocyclic compounds, 1,3-thiazole compounds, metalloprotease inhibitors, cyclosporin A, etc.) has been studied (for example, Non-Patent Documents 1 and 2). And Patent Documents 1 to 7).
TNF-α inhibitors are also known to exert an excellent preventive / therapeutic effect on pain, suppress osteoclast activation, and inhibit osteoclast formation. (For example, refer to Patent Document 6). Moreover, it is known that a TNF- (alpha) inhibitor can be used also for the treatment of glaucoma (for example, refer patent document 7).
白鶴霊芝（Rhinacanthus nasuta（L.）Kurz）は、インド南部デカン高原の原産とされるリナカンサス属きつねのまご科に属する常緑小低木であり、その全草（リナカンツス草）は駆虫、消炎、皮膚真菌に対する抗菌作用のあることが知られ（例えば、非特許文献３参照。）、主に中国、台湾等において、また最近では日本国においても漢方薬として用いられている。その他、本出願人による以前の出願で、白鶴霊芝に活性酸素消去能があること（特許文献８参照。）、排泄促進作用があること（特許文献９参照。）、抗アレルギー作用があること（特許文献１０参照。）および抗悪性腫瘍作用があること（特許文献１１参照。）が開示されている。しかしながら、この白鶴霊芝乃至白鶴霊芝の水および／または有機溶媒抽出物にＴＮＦ−α阻害作用があることは知られていない。 Rhinacanthus nasuta (L.) Kurz is an evergreen small shrub belonging to the genus Linakansus fox family that originated in the Deccan Plateau in southern India. It is known to have an antibacterial action against skin fungi (see, for example, Non-Patent Document 3), and is mainly used as a Chinese medicine in China, Taiwan, etc., and recently in Japan. In addition, in the previous application by the present applicant, Hakutsuru Ganoderma has active oxygen scavenging ability (see Patent Document 8), excretion promoting action (see Patent Document 9), and antiallergic action. (See Patent Document 10) and the fact that it has an anti-malignant tumor action (see Patent Document 11). However, it is not known that the water and / or organic solvent extract of this white crane reishi or white crane reishi has a TNF-α inhibitory action.
本発明者等は、ＴＮＦ−α阻害作用を有する物質につき鋭意研究の結果、従来漢方薬などとして使用されている白鶴霊芝の水および／または有機溶媒抽出物が有効であることを見出し、本発明を完成させるに至った。 As a result of intensive studies on substances having a TNF-α inhibitory effect, the present inventors have found that water and / or organic solvent extracts of white crane reishi used as traditional Chinese medicines are effective. It came to complete.
本発明は、白鶴霊芝の水および／または有機溶媒抽出物を主成分とすることを特徴とするＴＮＦ−α阻害剤である。 The present invention is a TNF-α inhibitor characterized by comprising water and / or an organic solvent extract of white crane reishi.
以下、本発明のＴＮＦ−α阻害剤を、発明を実施するための最良の形態に基づいてさらに詳細に説明する。 Hereinafter, the TNF-α inhibitor of the present invention will be described in more detail based on the best mode for carrying out the invention.
本発明のＴＮＦ−α阻害剤に使用する白鶴霊芝は、市販されている葉、全草、根もしくは茎、またはこれらの混合物を使用することができる。これらを天日乾燥および／またはドラム乾燥し、粉砕機にかけて１２メッシュ以上に粉砕して使用することも好ましい。白鶴霊芝は、必要により、天日乾燥および／またはドラム乾燥したものをさらに焙煎器により焙煎したものを使用することもできる。 As the white crane reishi used for the TNF-α inhibitor of the present invention, commercially available leaves, whole grass, roots or stems, or a mixture thereof can be used. It is also preferable that these are sun-dried and / or drum-dried and pulverized to 12 mesh or more by a pulverizer. If necessary, the white crane ganoderma can be used after being sun-dried and / or drum-dried and further roasted by a roaster.
本発明のＴＮＦ−α阻害剤は、このようにして得られた白鶴霊芝を水および／または有機溶媒で抽出して製造される。 The TNF-α inhibitor of the present invention is produced by extracting the white crane reishi obtained in this way with water and / or an organic solvent.
抽出に使用する溶媒としては、水および有機溶媒が挙げられ、これらを単独でまたは２種以上を混合して使用することができる。水には冷却水、常温水および熱水が包含される。 Examples of the solvent used for extraction include water and organic solvents, and these can be used alone or in admixture of two or more. Water includes cooling water, room temperature water and hot water.
抽出に使用する有機溶媒としては、エタノール、メタノール、プロパノール、１，３−ブチレングリコール等のアルコール類、ジメチルエーテル、ジエチルエーテル、メチルエチルエーテル等のエーテル類、アセトン等のケトン類、ベンゼン等のフェノール類、ヘキサン、ヘプタン、流動パラフィン、スクワラン等の炭化水素類等が挙げられるが、エタノールおよびメタノールが特に好ましい。 Examples of the organic solvent used for extraction include alcohols such as ethanol, methanol, propanol, and 1,3-butylene glycol, ethers such as dimethyl ether, diethyl ether, and methyl ethyl ether, ketones such as acetone, and phenols such as benzene. , Hydrocarbons such as hexane, heptane, liquid paraffin, squalane and the like, and ethanol and methanol are particularly preferable.
抽出は、粉砕した白鶴霊芝に５倍量から５０倍量の水または有機溶媒を加えて、放置、振とう、撹拌または還流しつつ行う。抽出は、０℃から１３０℃の温度で行うことができるが、室温から１１０℃の温度で行うことが好ましい。抽出に要する時間は、抽出温度および白鶴霊芝の粉砕状態にもよるが、通常３０分から６時間程度である。 Extraction is performed while adding 5 to 50 times the amount of water or an organic solvent to the ground white crane reishi and leaving, shaking, stirring or refluxing. The extraction can be performed at a temperature of 0 ° C. to 130 ° C., but is preferably performed at a temperature of room temperature to 110 ° C. The time required for extraction is usually about 30 minutes to 6 hours, although it depends on the extraction temperature and the ground state of white crane reishi.
抽出後、デカンテーシヨン、遠心分離、減圧濾過等の慣用手段を用いて、無臭乃至僅かな芳香、独特のコクのある味を帯びた透明で緑褐色乃至黒褐色の液体が得られる。これをそのまままたはさらに濾過、遠沈、その他の方法で精製したものを本発明のＴＮＦ−α阻害剤として使用することができる。
また、これを適度に濃縮して得られる濃縮液を本発明のＴＮＦ−α阻害剤として使用することもできる。
また、この濃縮液をさらに凍結乾燥、減圧濃縮乾固、スプレードライ、蒸留乾固等の乾燥手段で処理して得られる微粉末を本発明のＴＮＦ−α阻害剤として使用することもできる。
After the extraction, a transparent green-brown to black-brown liquid having an odorless or slightly fragrant and unique rich taste can be obtained by using conventional means such as decantation, centrifugation, and vacuum filtration. This can be used as the TNF-α inhibitor of the present invention as it is or after further purification by filtration, centrifugation, or other methods.
Moreover, the concentrate obtained by concentrating this moderately can also be used as the TNF-α inhibitor of the present invention.
Further, a fine powder obtained by further treating this concentrated solution with a drying means such as freeze drying, vacuum concentration drying, spray drying, distillation drying or the like can also be used as the TNF-α inhibitor of the present invention.
本発明のＴＮＦ−α阻害剤においては、上記した白鶴霊芝の水および／または有機溶媒抽出物に加えて、必要に応じて、本発明の効果を損なわない範囲で、食品、医薬部外品、医薬品等に一般に用いられる各種成分、すなわち水性成分、粉末成分、油分、界面活性剤、保湿剤、増粘剤、酸化防止剤、香料、色素等を配合することもできる。これにより、本発明のＴＮＦ−α阻害剤は、食品、医薬部外品、医薬品等の広範な用途に用いることができるようになる。 In the TNF-α inhibitor of the present invention, in addition to the water and / or organic solvent extract of Hakutsuru Reishi, the food and quasi-drug as long as the effects of the present invention are not impaired, if necessary. Various components generally used in pharmaceuticals, that is, an aqueous component, a powder component, an oil component, a surfactant, a moisturizer, a thickener, an antioxidant, a fragrance, a pigment, and the like can also be blended. Thereby, the TNF-α inhibitor of the present invention can be used for a wide range of applications such as foods, quasi drugs, and pharmaceuticals.
実施例１
天日乾燥し、次いでドラム乾燥した白鶴霊芝の根を粉砕機にて粉砕し、得られた粉砕物を篩にかけて１２メッシュで分級して白鶴霊芝根粉砕物を得た。この白鶴霊芝根粉砕物１ｋｇを９０％エタノール１０Ｌで３時間還流下に２回抽出し、得られた抽出液を５５℃〜６０℃で減圧下に約１０分の１量に濃縮後、加圧濾過した。濾液を９０℃〜９５℃で加熱滅菌後、減圧下に濃縮乾固し、微粉末状の白鶴霊芝根９０％エタノール抽出物８５．５ｇを得た。
Example 1
The roots of white crane reishi which were dried in the sun and then drum-dried were pulverized with a pulverizer, and the obtained pulverized product was sieved and classified with 12 mesh to obtain a pulverized white crane reishi. 1 kg of this white crane reishi mushroom pulverized product was extracted with 10 L of 90% ethanol twice under reflux for 3 hours, and the resulting extract was concentrated at about 55 to 60 ° C. under reduced pressure to about one-tenth volume. Pressure filtered. The filtrate was sterilized by heating at 90 ° C. to 95 ° C., and then concentrated to dryness under reduced pressure to obtain 85.5 g of a 90% ethanol extract of Hakutsukaku Reishi-ne root in fine powder form.
実施例２
天日乾燥し、次いでドラム乾燥した白鶴霊芝の葉を粉砕機にて粉砕し、焙煎器にて焙煎後、さらに粉砕し、得られた粉砕物を篩にかけて１２メッシュで分級して白鶴霊芝葉粉砕物を得た。この白鶴霊芝葉粉砕物１ｋｇを９０℃の熱水２５Ｌで２時間、２回抽出し、得られた抽出液を５５℃〜６０℃で減圧下に約１０分の１量に濃縮後、加圧濾過した。濾液を９０℃〜９５℃で加熱滅菌後、減圧下に濃縮乾固し、微粉末状の白鶴霊芝葉熱水抽出物３１０ｇを得た。
Example 2
White crane Ganoderma leaves dried in the sun and then drum dried are pulverized with a pulverizer, roasted with a roaster, further pulverized, and the resulting pulverized product is sieved and classified with 12 mesh. Reishiba crushed material was obtained. 1 kg of this white crane reishi mushroom pulverized product was extracted twice with 25 L of hot water at 90 ° C. for 2 hours, and the resulting extract was concentrated at about 55 ° C. to 60 ° C. under reduced pressure to about one-tenth amount. Pressure filtered. The filtrate was sterilized by heating at 90 ° C. to 95 ° C., and then concentrated to dryness under reduced pressure to obtain 310 g of a fine powdery white crane reishi leaf hot water extract.
実施例３
天日乾燥し、次いでドラム乾燥した白鶴霊芝の全草を粉砕機にて粉砕し、得られた粉砕物を篩にかけて１２メッシュで分級して白鶴霊芝全草粉砕物を得た。この白鶴霊芝全草粉砕物１ｋｇを９０℃の熱水２５Ｌで２時間、２回抽出し、得られた抽出液を５５℃〜６０℃で減圧下に約１０分の１量に濃縮後、加圧濾過した。濾液を９０℃〜９５℃で加熱滅菌後、減圧下に濃縮乾固し、微粉末状の白鶴霊芝全草熱水抽出物１８０ｇを得た。
Example 3
The whole white crane ganoderma dried in the sun and then drum-dried was pulverized with a pulverizer, and the obtained pulverized product was sieved and classified with 12 mesh to obtain a pulverized white crane ganoderma full grass. 1 kg of this white crane reishi mushroom pulverized product was extracted twice with 25 L of hot water of 90 ° C. for 2 hours, and the obtained extract was concentrated to about 1/10 of the reduced pressure at 55 ° C. to 60 ° C. Filtered under pressure. The filtrate was sterilized by heating at 90 ° C. to 95 ° C. and then concentrated to dryness under reduced pressure to obtain 180 g of Hakutsuru Reishi lawn whole plant hot water extract in the form of fine powder.
実施例４
実施例１と同様にして得られた白鶴霊芝根粉砕物２．５ｇを９０％エタノール１００ｍｌに浸漬、濾過して、僅かな芳香、独特のコクのある味を帯びた黒褐色の液状の白鶴霊芝根９０％エタノール抽出物を得た。
Example 4
2.5 g of white crane reishi mushroom pulverized product obtained in the same manner as in Example 1 was immersed in 100 ml of 90% ethanol and filtered to obtain a black brown liquid white crane with a slight aroma and a unique rich taste. Shibane 90% ethanol extract was obtained.
実施例５
実施例２と同様にして得られた白鶴霊芝葉粉砕物２．５ｇを９０℃の熱水１００ｍｌに浸漬、濾過して、僅かな芳香、独特のコクのある味を帯びた緑褐色の液状の白鶴霊芝葉熱水抽出物を得た。
Example 5
2.5 g of white crane reishi pulverized pulverized product obtained in the same manner as in Example 2 was immersed in 100 ml of hot water at 90 ° C. and filtered to give a greenish brown liquid with a slight aroma and a unique rich taste. The white crane Reishiba hot water extract was obtained.
実施例６
実施例３と同様にして得られた白鶴霊芝全草粉砕物２．５ｇを９０℃の熱水１００ｍｌに浸漬、濾過して、僅かな芳香、独特のコクのある味を帯びた深い緑色の液状の白鶴霊芝全草熱水抽出物を得た。
Example 6
2.5 g of crushed white crane reishi grass obtained in the same manner as in Example 3 was immersed in 100 ml of hot water at 90 ° C. and filtered to obtain a deep green color with a slight aroma and a unique rich taste. A liquid white crane reishi whole grass hot water extract was obtained.
試験例１
実施例１で得られた微粉末状の白鶴霊芝根９０％エタノール抽出物を用いてＴＮＦ−α阻害作用および毒性を評価した。
Test example 1
The TNF-α inhibitory action and toxicity were evaluated using the finely powdered white crane Reishi radish 90% ethanol extract obtained in Example 1.
Ａ．ＴＮＦ−α阻害作用の評価
１．肥満細胞の刺激
（１）抗原およびＩｇＥを用いた肥満細胞の刺激
ラットの肥満細胞（ＲＢＬ−２Ｈ３（ＪＣＲＢ Ｃｅｌｌ Ｂａｎｋ（細胞番号：ＪＣＲＢ００２３））(１×１０６ｃｅｌｌｓ／ｍｌ) を、マウス抗ＤＮＰ特異的ＩｇＥモノクローナル抗体にて１８時間感作した。次に、遠心分離後、Ｔｙｒｏｄｅ’ｓバッファー（０．０４％のＢＳＡおよび１０ｍＭのＨＥＰＥＳを含む。) により複数回洗浄した。次に、この細胞を９６ウェルプレートに入れ、表１に示す量のＤＮＰ−ＨＳＡ(human serum albumin)および微粉末状の白鶴霊芝根９０％エタノール抽出物を含む溶液を加えて２４時間刺激した。
A. Evaluation of TNF-α inhibitory action Stimulation of mast cells (1) Stimulation of mast cells using antigen and IgE Rat mast cells (RBL-2H3 (JCRB Cell Bank (cell number: JCRB0023)) (1 × 10 6 cells / ml) were treated with mouse anti-DNP. Sensitized with specific IgE monoclonal antibody for 18 hours, then centrifuged and washed multiple times with Tyrode's buffer (containing 0.04% BSA and 10 mM HEPES). Was added to a 96-well plate, and a solution containing DNP-HSA (human serum albumin) in an amount shown in Table 1 and a finely powdered white crane Reishi radish 90% ethanol extract was added and stimulated for 24 hours.
（２）イオノマイシンを用いた肥満細胞の刺激
ラットの肥満細胞（ＲＢＬ−２Ｈ３(１×１０６ｃｅｌｌｓ／ｍｌ) ）を９６ウェルプレートに入れ、表２に示す量のイオノマイシンおよび微粉末状の白鶴霊芝根９０％エタノール抽出物を加えて２４時間刺激した。
(2) Stimulation of mast cells using ionomycin Rat mast cells (RBL-2H3 (1 × 10 6 cells / ml)) were placed in a 96-well plate, and the amounts of ionomycin and finely powdered white crane spirit shown in Table 2 were used. Shibane 90% ethanol extract was added and stimulated for 24 hours.
２．ＴＮＦ−α阻害作用
（１）ＴＮＦ−α阻害作用の評価方法
ＴＮＦ−α阻害作用の評価は、上記１．の（１）および（２）のそれぞれについて、２４時間の刺激の後、上記した各９６ウェルプレートを遠心分離にかけ、上清を回収し、上清中のＴＮＦ−αの濃度をサンドイッチＥＬＩＳＡ法により測定することにより行った。サンドイッチＥＬＩＳＡ法による測定は、米国Ｒ＆Ｄシステムズ社（R&D Systems Inc.）のキットを用いて、付属のプロトコルに従って行った。その概要は以下の通りである。
2. TNF-α inhibitory action (1) Evaluation method of TNF-α inhibitory action For each of (1) and (2), after the stimulation for 24 hours, each 96-well plate described above was centrifuged, the supernatant was collected, and the concentration of TNF-α in the supernatant was determined by sandwich ELISA. This was done by measuring. The measurement by the sandwich ELISA method was performed using a kit of R & D Systems Inc. (USA) according to the attached protocol. The outline is as follows.
まず、あらかじめＴＮＦ−αに対するポリクローナル抗体でコートされたキット付属の９６ウェルマイクロプレートの各ウェルに、回収した上清を５０μｌ加え、室温で２時間反応させた。
次に、各ウェルの中の液体を除去し、キット付属の洗浄液により各ウェルを５回洗浄した後、キット付属の西洋ワサビペルオキシダーゼ（ＨＲＰ）が付加された、ＴＮＦ−αに対するモノクローナル抗体を含む液を各ウェルに１００μｌ加え、室温で２時間反応させた。
First, 50 μl of the collected supernatant was added to each well of a 96-well microplate attached to the kit previously coated with a polyclonal antibody against TNF-α, and reacted at room temperature for 2 hours.
Next, the liquid in each well is removed, each well is washed 5 times with the washing solution attached to the kit, and then a horseradish peroxidase (HRP) attached to the kit is added to the solution containing a monoclonal antibody against TNF-α. Was added to each well and allowed to react at room temperature for 2 hours.
次に、各ウェルの中の液体を除去し、キット付属の洗浄液により各ウェルを洗浄した後、キット付属の発色液を加えて室温で３０分間反応させ、その後、キット付属の反応停止液（hydrochloric acid）を加えて反応を停止させた。
得られた試料について、マイクロプレートリーダー（米国モレキュラーデバイス社（Molecular Device Corporation）製）を用いて、４５０ｎｍ（参照波長は５４０ｎｍ。）における吸光度を測定し、標準曲線からＴＮＦ−αの濃度を算出した。
Next, after removing the liquid in each well and washing each well with the cleaning solution included in the kit, add the coloring solution included in the kit and react at room temperature for 30 minutes. Then, the reaction stop solution (hydrochloric solution included in the kit) acid) was added to stop the reaction.
About the obtained sample, the light absorbency in 450 nm (reference wavelength is 540 nm) was measured using the microplate reader (Molecular Device Corporation (Molecular Device Corporation)), and the density | concentration of TNF- (alpha) was computed from the standard curve. .
（２）評価結果
評価結果を図１および図２に示す。図１は、上記１．の（１）（抗原およびＩｇＥを用いて肥満細胞を刺激した場合）における、肥満細胞からのＴＮＦ−αの放出量と白鶴霊芝根９０％エタノール抽出物の添加量との関係を示す図である。図２は、上記１．の（２）（イオノマイシンを用いて肥満細胞を刺激した場合）における、肥満細胞からのＴＮＦ−αの放出量と白鶴霊芝根９０％エタノール抽出物の添加量との関係を示す図である。
(2) Evaluation result An evaluation result is shown in FIG. 1 and FIG. FIG. (1) is a diagram showing the relationship between the amount of TNF-α released from mast cells and the amount of Hakutsuru Reishi-ne 90% ethanol extract added (when mast cells are stimulated with antigen and IgE) is there. FIG. It is a figure which shows the relationship between the release amount of TNF- (alpha) from a mast cell, and the addition amount of a white crane reishi root 90% ethanol extract in (2) (when a mast cell is stimulated using ionomycin).
図１および図２に示すように、実施例１における白鶴霊芝根９０％エタノール抽出物は、優れたＴＮＦ−α阻害作用を有することがわかった。 As shown in FIG. 1 and FIG. 2, it was found that the 90% ethanol extract of Hakutsukaku Reishi root in Example 1 has an excellent TNF-α inhibitory action.
３．免疫抑制剤シクロスポリンＡとの比較
白鶴霊芝根９０％エタノール抽出物のＴＮＦ−α阻害作用と、免疫抑制剤シクロスポリンＡのＴＮＦ−α阻害作用との比較を行った。比較は、表３に示す量のＤＮＰ−ＨＳＡ(human serum albumin)を用いた場合について行った。
3. Comparison with Immunosuppressant Cyclosporin A The TNF-α inhibitory action of Hakutsuru Reishi-ne 90% ethanol extract was compared with the TNF-α inhibitory action of the immunosuppressant cyclosporin A. The comparison was performed when the amount of DNP-HSA (human serum albumin) shown in Table 3 was used.
図３は、白鶴霊芝のＴＮＦ−α阻害作用と免疫抑制剤シクロスポリンＡのＴＮＦ−α阻害作用との関係を示す図である。
図３に示すように、実施例１における白鶴霊芝根９０％エタノール抽出物は、免疫抑制剤シクロスポリンＡと同様に優れたＴＮＦ−α阻害作用を有することがわかった。
FIG. 3 is a diagram showing the relationship between the TNF-α inhibitory action of white crane reishi and the TNF-α inhibitory action of the immunosuppressant cyclosporin A.
As shown in FIG. 3, it was found that the white crane reishi mushroom 90% ethanol extract in Example 1 has an excellent TNF-α inhibitory action similar to the immunosuppressant cyclosporin A.
Ｂ．毒性の評価
毒性の評価は、ラットの肥満細胞（ＲＢＬ−２Ｈ３)に表４に示す濃度の白鶴霊芝根９０％エタノール抽出物を添加して２４時間の培養を行った後、ＭＴＳ試薬を加えさらに２時間の培養を行い、その後、マイクロプレートリーダー（テカン ジャパン社製）を用いて４９０ｎｍの吸光度を測定することにより行った。
B. Toxicity evaluation Toxicity evaluation was performed by adding 90% ethanol extract of Hakutsuru Reishi-ne root with the concentrations shown in Table 4 to rat mast cells (RBL-2H3), followed by incubation for 24 hours, and then adding MTS reagent. The culture was further performed for 2 hours, and then the absorbance at 490 nm was measured using a microplate reader (Tecan Japan).
その結果、試料Ｎｏ．１乃至試料Ｎｏ．５におけるＭＴＳ結果から、白鶴霊芝根９０％エタノール抽出物は、肥満細胞に対する毒性が極めて低いことがわかった。 As a result, sample no. 1 to Sample No. From the MTS results in Fig. 5, it was found that the white crane Reishi mushroom 90% ethanol extract has extremely low toxicity to mast cells.
Ｃ．試験例１のまとめ
以上より、白鶴霊芝根９０％エタノール抽出物は、優れたＴＮＦ−α阻害作用および高い安全性を有する、優れたＴＮＦ−α阻害剤であることがわかった。
C. From the summary of Test Example 1, it was found that the white crane reishi mushroom 90% ethanol extract is an excellent TNF-α inhibitor having an excellent TNF-α inhibitory action and high safety.
試験例２
実施例１で得られた白鶴霊芝根９０％エタノール抽出物に代えて、実施例２で得られた白鶴霊芝葉熱水抽出物を用いたこと以外は、試験例１と同様にして、ＴＮＦ−α阻害作用および毒性を評価した。
その結果、実施例２で得られた白鶴霊芝葉熱水抽出物は、実施例１の場合と同様に、優れたＴＮＦ−α阻害作用および高い安全性を有することがわかった。
Test example 2
In the same manner as in Test Example 1, except that the white crane reishi leaf hot water extract obtained in Example 2 was used instead of the white crane reishi root 90% ethanol extract obtained in Example 1, TNF-α inhibitory action and toxicity were evaluated.
As a result, it was found that the white crane reishi leaf hot water extract obtained in Example 2 had an excellent TNF-α inhibitory action and high safety, as in Example 1.
試験例３
実施例１で得られた白鶴霊芝根９０％エタノール抽出物に代えて、実施例３で得られた白鶴霊芝全草熱水抽出物を用いたこと以外は、試験例１と同様にして、ＴＮＦ−α阻害作用および毒性を評価した。
その結果、実施例３で得られた白鶴霊芝全草熱水抽出物は、実施例１の場合と同様に、優れたＴＮＦ−α阻害作用および高い安全性を有することがわかった。
Test example 3
The same procedure as in Test Example 1 was used except that the white crane reishi mushroom whole grass hot water extract obtained in Example 3 was used in place of the 90% ethanol extract of white crane reishi mushroom obtained in Example 1. The TNF-α inhibitory action and toxicity were evaluated.
As a result, it was found that the white crane reishi whole grass hot water extract obtained in Example 3 had an excellent TNF-α inhibitory action and high safety as in Example 1.
試験例４
実施例１で得られた白鶴霊芝根９０％エタノール抽出物に代えて、実施例４で得られた白鶴霊芝根９０％エタノール抽出液を用いたこと以外は、試験例１と同様にして、ＴＮＦ−α阻害作用および毒性を評価した。
その結果、実施例４で得られた白鶴霊芝根９０％エタノール抽出液は、実施例１の場合と同様に、優れたＴＮＦ−α阻害作用および高い安全性を有することがわかった。
Test example 4
The same procedure as in Test Example 1 was used except that the white crane reishi mushroom 90% ethanol extract obtained in Example 1 was used instead of the white crane reishi mushroom 90% ethanol extract obtained in Example 4. The TNF-α inhibitory action and toxicity were evaluated.
As a result, it was found that the white crane reishi mushroom 90% ethanol extract obtained in Example 4 had an excellent TNF-α inhibitory action and high safety as in Example 1.
試験例５
実施例１で得られた白鶴霊芝根９０％エタノール抽出物に代えて、実施例５で得られた白鶴霊芝葉熱水抽出液を用いたこと以外は、実施例１と同様にして、ＴＮＦ−α阻害作用および毒性を評価した。
その結果、実施例５で得られた白鶴霊芝葉熱水抽出液は、実施例１の場合と同様に、優れたＴＮＦ−α阻害作用および高い安全性を有することがわかった。
Test Example 5
In the same manner as in Example 1 except that the white crane reishi leaf hot water extract obtained in Example 5 was used instead of the white crane reishi turf root 90% ethanol extract obtained in Example 1, TNF-α inhibitory action and toxicity were evaluated.
As a result, it was found that the white crane ganoderma hot water extract obtained in Example 5 had an excellent TNF-α inhibitory action and high safety as in Example 1.
試験例６
実施例１で得られた白鶴霊芝根９０％エタノール抽出物に代えて、実施例６で得られた白鶴霊芝全草熱水抽出液を用いたこと以外は、実施例１と同様にして、ＴＮＦ−α阻害作用を評価した。
その結果、実施例６で得られた白鶴霊芝全草熱水抽出液は、実施例１の場合と同様に、優れたＴＮＦ−α阻害作用および高い安全性を有することがわかった。
Test Example 6
The same procedure as in Example 1 was used except that the white crane reishi mushroom whole grass hot water extract obtained in Example 6 was used in place of the 90% ethanol extract of white crane reishi root obtained in Example 1. The TNF-α inhibitory action was evaluated.
As a result, it was found that the white crane reishi whole grass hot water extract obtained in Example 6 had an excellent TNF-α inhibitory action and high safety as in Example 1.
以上説明したように、本発明のＴＮＦ−α阻害剤は、優れたＴＮＦ−α阻害作用を有するため、ＴＮＦ−αの過剰な産生が引き起こす組織障害やさまざまな病気（例えば、関節リウマチ、全身性エリテマトーデス（ＳＬＥ）、悪液質、急性感染症、アレルギー、発熱、貧血、糖尿病など。）の治療および予防効果が期待できる。また、本発明のＴＮＦ−α阻害剤は、高い安全性を有するため、長期連用しても人体に有害な作用を及ぼさない利益がある。 As described above, since the TNF-α inhibitor of the present invention has an excellent TNF-α inhibitory action, tissue damage caused by excessive production of TNF-α and various diseases (for example, rheumatoid arthritis, systemic) Therapeutic and preventive effects of lupus erythematosus (SLE), cachexia, acute infection, allergy, fever, anemia, diabetes, etc.) can be expected. In addition, since the TNF-α inhibitor of the present invention has high safety, there is a benefit that it does not have a harmful effect on the human body even if used for a long time.
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| JP2009534419A (en) * | 2006-04-21 | 2009-09-24 | セントカー・インコーポレーテツド | CXCL13 antagonists and their use for the treatment of inflammatory diseases |
| CN104706950A (en) * | 2015-03-03 | 2015-06-17 | 广西大学 | Soup capable of improving female anemia and preparation method for soup |
| JP2023028872A (en) * | 2021-08-20 | 2023-03-03 | 株式会社アルソア慧央グループ | METHOD FOR PRODUCING AGING-RELATED IL-1β BLOOD CONCENTRATION RISE INHIBITOR FOR ORAL INGESTION |
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| JP2001010964A (en) * | 1999-06-25 | 2001-01-16 | Arsoa Honsya Corp | Antiallergic agent |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2009534419A (en) * | 2006-04-21 | 2009-09-24 | セントカー・インコーポレーテツド | CXCL13 antagonists and their use for the treatment of inflammatory diseases |
| CN104706950A (en) * | 2015-03-03 | 2015-06-17 | 广西大学 | Soup capable of improving female anemia and preparation method for soup |
| JP2023028872A (en) * | 2021-08-20 | 2023-03-03 | 株式会社アルソア慧央グループ | METHOD FOR PRODUCING AGING-RELATED IL-1β BLOOD CONCENTRATION RISE INHIBITOR FOR ORAL INGESTION |
| JP7239208B2 (en) | 2021-08-20 | 2023-03-14 | 株式会社アルソア慧央グループ | Method for producing aging-related IL-1β blood level elevation inhibitor for oral intake |
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